Identification of a Prothoracicostatic Peptide in the Larval Brain of the Silkworm, Bombyx mori

Yue-Jin Hua,Yoshiaki Tanaka,Keiji Nakamura,Mika Sakakibara,Shinji Nagata,Hiroshi Kataoka

doi:10.1074/jbc.274.44.31169

Abstract

Prothoracicotropic hormone (PTTH) stimulates ecdysteroid biosynthesis in the prothoracic gland (PG) of insects. A peptide inhibiting ecdysteroid biosynthesis in the PG was isolated from the extracts of 2,000 larval brains of the silkworm, Bombyx mori, using a protocol that included four reversed-phase high performance liquid chromatography procedures. The primary structure of this prothoracicostatic peptide (Bom-PTSP) was determined to be H-Ala-Trp-Gln-Asp-Leu-Asn-Ser-Ala-Trp-NH(2). This neuropeptide has the same sequence as Mas-MIP-I, a myoinhibitory peptide previously isolated from the ventral nerve cord of the tobacco hornworm, Manduca sexta, and is highly homologous with the N-terminal portion of vertebrate peptides of the galanin family. This peptide inhibited PTTH-stimulated ecdysteroidogenesis in the PG at both the spinning and feeding stages, which indicates that Bom-PTSP interferes with PTTH-stimulated ecdysteroidogenesis.

Highlights

Prothoracicotropic hormone (PTTH) stimulates ecdysteroid biosynthesis in the prothoracic gland (PG) of insects
Purification of Bom-prothoracicostatic peptide (PTSP)—Fractions that had been eluted with 35% acetonitrile from the C18 Sep-Pak cartridge showed strong prothoracicostatic activity in the in vitro bioassay, which was completely eliminated by Pronase treatment; this indicated that the biological material is a peptide
The activation ratio (Ar) of PGs that had been incubated in media that contained 10Ϫ10 M PTTH, and the indicated concentration of Bom-PTSP was significantly lower than the Ar of age-matched PGs that had been incubated in the presence of PTTH alone

Summary

EXPERIMENTAL PROCEDURES

A racial hybrid, C145 ϫ N140, of B. mori was used. Larvae were reared at 25 Ϯ 1 °C under a photoperiodic regime of 12 h of light and 12 h of darkness on a standard artificial diet Prothoracicostatic activity was detected in the 35% acetonitrile fraction Four of these Sep-Pak separations were required to process all of the brain material. The 35% acetonitrile Sep-Pak fraction (2,000 brain equivalents) was evaporated to reduce the concentration of acetonitrile, diluted 5-fold with aqueous 0.1% TFA and loaded onto the Pegasil300 column through a pump. Pooled Fraction 29 from the first HPLC separation was dried to a small volume, dissolved in 500 ␮l of 0.1% TFA in water, and loaded onto the second column. Fraction 11 from the second HPLC separation was dried to a small volume, dissolved in 500 ␮l of 0.1% TFA in water, and loaded onto the third column. Prothoracicostatic material from the third step (the fraction between 24.6 and 25.8 min) was dried to a small volume, dissolved in 500 ␮l of 0.1% TFA in water and loaded onto the C4 column. One of the pair of PGs of a larva was incubated in medium containing various concentrations of PTTH and synthetic Bom-PTSP peptide, whereas the other PG was incubated in medium that contained neither PTTH nor Bom-PTSP to study the effect of combined PTTH and Bom-PTSP treatment on ecdysteroidogenesis

RESULTS

1.30 Ϯ 0591a

DISCUSSION